by High-performance liquid chromatography (HPLC) is an analytical technique to separate, identify, and quantify components in a mixture. It is the single biggest chromatography technique essential to most laboratories worldwide.
How Does HPLC Work?
The solvent used to separate components in a liquid sample for HPLC analysis is called the mobile phase. The mobile phase is delivered to a separation column, otherwise known as the stationary phase, and then to the detector at a stable flow rate controlled by the solvent delivery pump. A certain amount of sample is injected into the column and the compounds contained in the sample are separated. The compounds separated in the column are detected by a detector downstream of the column and each compound is identified and quantified.
In column chromatography a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography. A pump forces a solvent through a column under high pressures of up to 400 atmospheres. The column packing material or adsorbent or stationary phase is typically a granular material made of solid particles such as silica or polymers.
The pressure makes the technique much faster compared to column chromatography. This allows using much smaller particles for the column packing material. The smaller particles have a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This results in a much better separation of the components of the mixture.
The pressurized liquid is typically a mixture of solvents such as water, acetonitrile and/or methanol and is referred to as the mobile phase.
The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column. Compared to column chromatography.
HPLC Separation
HPLC can separate and detect each compound by the difference of each compound’s speed through the column.
There are two phases for HPLC: the mobile phase and the stationary phase. The mobile phase is the liquid that dissolves the target compound. The stationary phase is the part of a column that interacts with the target compound.
Mobile Phase
The solvent used to separate components in a liquid sample for HPLC analysis is called the mobile phase. The mobile phase is delivered to a separation column, otherwise known as the stationary phase, and then to the detector at a stable flow rate controlled by the solvent delivery pump.
Stationary phase
The stationary phase is the part of a column that interacts with the target compound. In the column, the stronger the affinity between the component and the mobile phase, the faster the component moves through the column along with the mobile phase.
Types of HPLC
The two most common variants are normal-phase and reversed-phase HPLC.
Normal-Phase HPLC
The column is filled with tiny silica particles, and a non-polar solvent, for example, hexane. Non-polar compounds in the mixture will pass more quickly through the column, as polar compounds will stick longer to the polar silica than non-polar compounds will.
Reversed-Phase HPLC
The column is filled with silica particles which are modified to make them non-polar. This is done by attaching long hydrocarbon chains (8–18 C atoms) to its surface. A polar solvent is used, for example, a mixture of water and an alcohol such as methanol. Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture.
Non-polar molecules are slowed down on their way through the column. They form varying degrees of attraction with the hydrocarbon groups principally through van der Waals dispersion forces and hydrophobic interactions. They are also less soluble in the aqueous mobile phase components facilitating their interactions with the hydrocarbon groups.
Reversed phase HPLC is the most commonly used form of HPLC
HPLC Apparatus
HPLC consists of a variety of components, including a solvent delivery pump, a degassing unit, a sample injector, a column oven, a detector, and a data processor etc. HPLC flow diagram and the role of each component.
